Interleukin-32 (IL-32) was initially identified in 1992 by Dahl et al. as a cytokine-like molecule named natural killer cell transcript 4 (NK4). Since its initial identification in 1992, NK4 has not been extensively studied and the term NK4 (or NK-4) was assigned also to other unrelated proteins [Kim, 1989; Smart, 1989; Date, 1997]).
Increased gene expression of NK4 in peripheral blood mononuclear cells (PBMC) from patients receiving high-dose IL-2 therapy for malignant melanoma has been reported, but its function has not been determined [Panelli, 2002].
It has been recently reported by Kim et al. (2005) that stimulation of Raw 264.7 macrophage cells with recombinant NK4 induced secretion of large amounts of TNF-α in these cells. Therefore, NK4 was recognized as a pro-inflammatory cytokine and was renamed to IL-32 [Kim, 2005].
The gene encoding IL-32 resides in the human chromosome 16 p13.3. The IL-32 gene contains eight exons. Four IL-32 mRNA splice variant encoding IL-32α, IL-32β, L-32δ and IL-32γ isoforms, were detected in human natural killer (NK) cells, and the IL-32γ isoform was identified to be identical to the transcript previously reported as the NK4 transcript [Kim, 2005]. The IL-32γ transcript, including exons 3 and 4, encodes a protein isoform having 46 additional amino acids at the N terminus. In the IL-32δ transcript the second exon is absent and initiation of translation occurs at an ATG codon present at the third exon. Unlike the other variants, the IL-32α transcript lacks exons 7 and 8 and encodes a protein isoform missing 57 amino acid residues at its C terminus. Out of the four IL-32 transcripts, the IL-32α transcript is the most abundant, hence IL-32α was more extensively characterized. Analysis of the amino acid sequence of the IL-32α isoform revealed three potential N-myristoylation sites and one N-glycosylation site [Kim, 2005].
IL-32α and β were reported to induce secretion of significant amounts of tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-2 (MIP-2) in a dose-dependent manner from PMA-differentiated THP-1 cells and from mouse Raw cells. Recombinant IL-32-α and β (rIL-32-α and β) were reported to induce IL-8 production in non-differentiated human monocytic THP-1 cells. An Fab fragment of a monoclonal antibody directed against rIL-32β was found to reduce the biological activity of rIL-32α by up to 70% in a dose-dependent manner. At the transcriptional level, the 1.2 KB IL-32 mRNA was detected in several tissues, but was more prominent in immune cells than in non-immune tissues (Kim, 2005).
Human peripheral blood mononuclear cells (PBMC), which contain mostly T cells, produced and secreted IL-32 following stimulation with Con A (inducing mainly T cell stimulation). Production or secretion of IL-32 was not detected following lipopolysaccharides stimulation of PBMC (inducing mainly macrophage stimulation). These results suggest that T cells are the major producers of IL-32. Nevertheless, stimulation of epithelial cell lines with IFN-gamma was found to induce IL-32 production.
The pro inflammatory activity of IL-32 appears to be mediated through degradation of I-κB, leading to activation of NF-κB. However, MAP kinase activation by IL-32α has also been reported [Kim, 2005].
Proteinase 3 (PR-3, also known as myeloblastin, neutrophil PR-3 and Wegener autoantigen) is a granule serine protease produced by neutrophiles/monocytes and is capable of processing multiple biologic substrates [Baggiolini, 1978; Kao, 1988]. PR-3 degrades a variety of extracellular matrix proteins, including elastin, fibronectin, type IV collagen, and laminin and inactivates p65 NF-κB [Preston, 2002]. PR-3 cleaves many pro-hormones and cytokines including angiotensinogen, TGF-β1, IL-1β, IL-8 and the membrane bound TNF-α into their active form [Ramaha, 2002; Csernok, 1996; Coeshott, 1999; Padrines, 1994; Robache-Gallea, 1995]. Indeed, high titers of PR-3 autoantibodies (see below) were found to completely block the cleavage of TNF-α.
PR-3, which appears both in soluble and cell membrane forms, is the major autoantigen in Wegener's granulomatosis (WG). Wegener's granulomatosis is the most common autoimmune necrotizing systemic vasculitis in adults and is manifested mainly in the respiratory tract and kidneys [Lamprecht, 2004; Frosch, 2004].
Autoantibodies to PR-3, known as “Anti-neutrophil cytoplasmic autoantibodies” (ANCA) are a diagnostic hallmark of WG [van Rossum, 2003; Jennette, 1997]. The frequency of the membrane PR-3 (mPR-3)-high phenotype was found to be significantly higher in patients with ANCA-associated vasculitis and in patients with rheumatoid arthritis. Hence, membrane PR-3 expression is a risk factor for vasculitis and rheumatoid arthritis [Witko-Sarsat, 1999]. Expression of PR-3 in the membranes of neutrophil cells is related to relapse in PR-3-anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Patients with small vessel vasculitis have increased levels of circulating PR-3 protein in their plasma [Ohlsson, 2003]. High levels of PR-3 expression on the membrane of neutrophils is also a WG risk factor and is associated with relapse of WG disease (Rarok A A, Stegeman C A, Limburg P C, Kallenberg C G. J Am Soc Nephrol. 2002 September; 13(9): 2232-8.
Apparently, pathogenesis of WG is induced by the binding of ANCA to PR-3 antigen present on the surface of neutrophils and monocytes. Binding of ANCA to neutrophils and monocytes causes cell activation, respiratory burst and release of toxic oxygen radicals and proteolytic enzymes. The exposure of PR-3 on the cell surface and binding of anti-PR-3 autoantibodies to neutrophils appears to facilitate autoimmunization and amplification of neutrophil-induced vascular inflammation.
Gene expression profiles of peripheral mature neutrophils and monocytes from patients suffering from ANCA diseases manifested in the kidney showed increased levels of transcripts of a group of genes that are normally expressed only in bone marrow precursor cells (“left shift”). PR-3 transcript is included in this group of increased genes and the increase of PR-3 expression correlated with the disease activity and with glomerulonephritis [Muller Kobold, 1998; Yang, 2004; Yang, 2002].
Cystic fibrosis (CF) patients have increased PR-3 mRNA in circulating monocytes at the time of pulmonary exacerbation [Just, 1999]. Surfactant protein D (SP-D) is an important innate host defense molecule present in the lung of CF affected patients, which interacts with CF-associated pathogens [von Bredow, 2003]. SP-D is a target protein for PR-3. Thus, in CF patients the host defense appears to be impaired due to proteolysis of SP-D by PR-3, thereby increasing the incidence of infection of the lung in these patients.
In patients with inflamed gums functional PR-3 was found to be expressed in oral epithelial cells and ANCA was found in the patient's serum. Said epithelial cells expressing functional PR-3 appear to participate in the inflammatory processes of the gums, including gingivitis and periodontitis [Uehara, 2004].
Besides acting on the cell surface and in the extracellular space, PR-3 enters endothelial cells, where it can mimic caspases, for example, by cleaving NF-κB and inducing sustained JNK activation. PR-3 also cleaves and inactivates the major cell cycle inhibitor p21Waf1/Cip1/Sdi1. High levels of PR-3 and p21 cleavage product were found in inflamed human tissue taken from Crohn's disease patients and from ulcerative colitis [Pendergraft, 2004].
Dipeptidyl peptidase I (DPPI) is required for the full activation of neutrophil derived serine proteases such as PR-3. PR-3 knockout mice are not available, but DPPI-deficient mice were successfully generated [Adkison, 2002]. The DPPI knockout mice were found to be resistant to arthritis induction by anti-collagen antibodies and did not accumulated neutrophils in their joints. Resistance to arthritis induction correlated with inactivation of neutrophil-derived serine proteases since knockout mice deficient in serine proteases such as neutrophil elastase (−/−)×cathepsin G (−/−) were shown to be also resistant to induction of arthritis by anti-collagen antibodies.
Enzymatically inactive PR-3 fragments generated by deletion of the catalytic triad [Yang, 2001] still maintain several biological activities, including:                (i) down-modulation of DNA synthesis in normal hematopoietic progenitor cells, an effect which can be reversed by granulocyte-macrophage colony stimulating factor (GM-CSF), implying that PR-3 can function as a counterbalance to regulators of proliferation [Skold, 1999].        (ii) Induction of interleukin-8, both at transcriptional and translational levels [Berger, 1996], and        (iii) Induction of apoptosis in human umbilical vein endothelial cells (HUVEC) [Yang, 2001].        
As previously mentioned, PR-3 is a serine proteinase and many well-characterized natural and synthetic serine proteinase inhibitors are capable of inactivating PR-3, either reversibly or irreversibly. Several serine proteinase inhibitors were reported to specifically inhibit PR-3. The synthetic inhibitors 7-amino-4-chloro-3-(2-bromoethoxy) isocoumarin and 3,4-dichloroisocoumarin (DCI) exhibited kI values of 4700 and 2600 M−1·s−1, respectively [Kam, 1992]. Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and PR-3. The Ki for PR-3 is 5·10−7 M [Cadene, 1997]. A general class of peptidomimetic agents based on 1,2,5-thiadiazolidin-3-one 1,1-dioxide backbone was described and their sulfone derivatives were found to be time-dependent, potent, and highly efficient irreversible inhibitors of human leukocyte elastase, cathepsin G, and PR-3 [Groutas, 1997]. Such compounds were found to be useful as anti-inflammatory agents (Groutas W C., U.S. Pat. No. 5,550,139 Aug. 27, 1996).
Other proteinase inhibitors consist of polypeptides of various sources. Elafin, a human skin derived peptide that inhibits human leukocyte elastase, was shown to be a potent inhibitor of PR-3, showing an IC50 of 9.5×10−9 M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C [Wiedow, 1991; Zani, 2004]. MNEI (monocyte/neutrophile elastase inhibitor) is a 42 kDa serpin superfamily member, which efficiently inhibits proteases with elastase- and chymotrypsin-like specificities. MNEI rapidly inhibited PR-3 at a rate >107 M−1·s−1 [Cooley, 2001]. A bioengineered serpin (LEX032) was found to be a time-dependent inhibitor of PR-3, forming a highly stable enzyme-inhibitor complex (Ki 12 nM) [Groutas, 1997]. Thus, many serine proteinase inhibitors were specifically shown to inhibit PR-3.